Liraglutide attenuates high glucose-induced abnormal cell migration, proliferation, and apoptosis of vascular smooth muscle cells by activating the GLP-1 receptor, and inhibiting ERK1/2 and PI3K/Akt signaling pathways

Background As a new anti-diabetic medicine, Liraglutide (LIRA), one of GLP-1 analogues, has been found to have an anti-atherosclerotic effect. Since vascular smooth muscle cells (VSMCs) play pivotal roles in the occurrence of diabetic atherosclerosis, it is important to investigate the role of LIRA in reducing the harmful effects of high-glucose (HG) treatment in cultured VSMCs, and identifying associated molecular mechanisms. Methods Primary rat VSMCs were exposed to low or high glucose-containing medium with or without LIRA. They were challenged with HG in the presence of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK)1/2, or glucagon-like peptide receptor (GLP-1R) inhibitors. Cell proliferation and viability was evaluated using a Cell Counting Kit-8. Cell migration was determined by Transwell migration and scratch wound assays. Flow cytometry and Western blotting were used to determine apoptosis and protein expression, respectively. Results Under the HG treatment, VSMCs exhibited increased migration, proliferation, and phosphorylation of protein kinase B (Akt) and ERK1/2, along with reduced apoptosis (all p < 0.01 vs. control). These effects were significantly attenuated with LIRA co-treatment (all p < 0.05 vs. HG alone). Inhibition of PI3K kinase and ERK1/2 similarly attenuated the HG-induced effects (all p < 0.01 vs. HG alone). GLP-1R inhibitors effectively reversed the beneficial effects of LIRA on HG treatment (all p < 0.05). Conclusions HG treatment may induce abnormal phenotypes in VSMCs via PI3K and ERK1/2 signaling pathways activated by GLP-1R, and LIRA may protect cells from HG damage by acting on these same pathways.


Background
Cardiovascular disease remains the leading cause of mortality and morbidity in diabetes mellitus (DM) patients [1,2]. Atherosclerosis is a complication that can be triggered by damage to vascular smooth muscle cells (VSMCs) in DM patients [3]. High glucose (HG) levels in the blood of DM patients often results in an enhanced generation of reactive oxygen species products, which can stimulate the proliferation and migration of VSMCs, causing accumulation of VSMCs in the intima of blood vessels [4,5]. Additionally, abnormal apoptosis of VSMCs has been observed in atherosclerosis and other DM-related cardiovascular diseases [6]. In DM patients, HG inhibits apoptosis of VSMCs by upregulating anti-apoptotic proteins, including Bcl-2, Bcl-xL, and Bfl-1/A1 [7,8]. Overproliferation or reduced apoptosis of VSMCs accelerates the deposition of atherosclerotic plaques in the lining of blood vessels, and induces intimal thickening and vascular remodeling [9]. Strategies preventing HG-induced alterations to VSMC cell migration, proliferation, and apoptosis may represent promising therapies for protecting blood vessels against diabetic atherosclerosis.
Glucagon-like peptide-1 (GLP-1), a gut incretin, modulates glucose-dependent insulin secretion and suppresses the release of glucagon [24]. A large body of evidence indicates that GLP-1 plays an important role in the pathogenesis of diabetic atherosclerosis. Long-term treatment with GLP-1 effectively improves severe obesity, hypertension, and lipid profiles, all of which are critical risk factors in the development of atherosclerosis [25][26][27][28]. GLP-1 also has multiple therapeutic effects on the cardiovascular system, improving cardiac function and exerting direct protective effects on cardiomyocytes [29][30][31], endothelial cells [32,33], macrophages [34][35][36], and VSMCs [37]. Moreover, animal studies have demonstrated that GLP-1 can significantly inhibit atherosclerotic plaque deposition in arteries, the formation of macrophage-derived foam cells and the adhesion of mononuclear cells in the intima, and attenuate the abnormal expression of CD36 [34,38]. It also prevents vascular remodeling and protects endothelial cells against oxidative stress via ameliorating intima inflammatory reactions [24,39,40].
Although the molecular mechanisms responsible for the effects of GLP-1 in the cardiovascular system are still uncertain, anti-apoptotic effects of GLP-1 on cardiomyocytes involve regulation of the PI3K/Akt and ERK1/2 signaling pathways [31,[41][42][43]. Furthermore, GLP-1 affects human endothelial cell proliferation through phosphorylation of Akt [44]. As these PI3K/Akt and ERK1/2 signaling pathways are also involved in the effects of HG on VSMCs [13][14][15]19,20,22,23], we hypothesized that they are responsible for the effects of GLP-1 on VSMCs treated with HG.
GLP-1 specifically binds to GLP-1 receptor (GLP-1R) to stimulate the adenylyl cyclase pathway resulting in increased insulin synthesis and release [45,46]. GLP-1R is expressed on VSMCs [47], and platelet-derived growth factor-induced VSMC cell proliferation is significantly inhibited by a GLP-1R agonist (Exendin-4) [48]. However, no efforts have been made to examine the direct effects of GLP-1 on the HG-induced cell migration, proliferation, and apoptosis of cultured VSMCs.
In this study, we investigated the role of liraglutide (LIRA), a GLP-1 analog, in the attenuation of HG-induced VSMC migration, proliferation, and reduced apoptosis. Furthermore, the mechanisms underlying these effects were also studied.

Animals
Male Sprague Dawley rats (n = 4; 5-8 wks) were provided by the Laboratory Animal Center of Harbin Medical University, China. All procedures were performed in accordance with guidelines set by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Harbin Medical University, which is in compliance with the ARRIVE guidelines on animal research [49].

Cell culture
VSMCs were prepared from thoracic aorta of Sprague Dawley rats as previously described, with minor modifications [50]. Each rat was sacrificed and the whole thoracic aorta was isolated and washed several times in phosphate buffered saline (PBS). After the adventitia and intima were carefully removed, the aortic tissue was cut into small pieces (1 mm 2 ). Theses explants were plated into a tissue culture flask and cultured in DMEM supplemented with 15% FBS and maintained in a humidified incubator at 37°C and 5% CO 2 . The cells were passaged by trypsinization and reseeded into new flasks approximately 4-8 times before use in subsequent experiments. VSMCs were identified by α-SMA staining.

Cell proliferation assay
Cells were plated in 96-well culture plates and incubated until reaching 80% confluency; the culture media was then replaced with serum-free DMEM and incubated for an additional 24 h. Cells were then treated with the media containing the indicated concentration of various purposely designed chemical(s) for an additional 48 h. Cell proliferation was then assessed using a Cell Counting Kit-8, following the manufacturer's instructions. Briefly, the colorimetric reagent, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt, was added to each sample and incubated for 1 h at 37°C. Proliferation was then assessed by measuring absorbance of each sample at the wavelength of 450 nm.

Transwell migration assay
Cell migration was determined using the Transwell migration assay as previous described, with minor modification [56]. Briefly, VSMCs were treated with the indicated chemical(s) for 12 h at 37°C. The cells were washed three times with PBS after the culture media was removed and then trypsinized with 0.25% (v/v) trypsin and resuspended in serum-free DMEM at 37°C. These cells were then counted and the upper chamber of each Transwell was seeded with 1 × 10 5 cells per chamber in 0.2 mL serum-free DMEM. As a chemoattractant, 0.8 mL of DMEM supplemented with 20% FBS was added to the lower chamber of each Transwell. Chambers were incubated for 12 h at 37°C with 5% CO 2 . Cells that migrated to the underside of the Transwell filter were fixed with 4% formaldehyde (w/v) for 20 min at room temperature and then immersed into a hematoxylin staining solution for 15 min. After washing with distilled water, membranes were mounted on glass slides and examined by microscopy at 200× magnification.

In vitro scratch wound assay
The migration capacity of VSMCs was also characterized using a well-established in vitro scratch wound model, with minor modifications [57]. VSMCs were grown to confluence and then subjected to scratching using a 200 μL sterile pipette tip. The scratch wound was allowed to heal for 24 h in the presence of the indicated chemical(s). Micrographs were captured for each sample at 0 and 24 h, and the capacity of VSMC migration was evaluated by measuring the width of the scratch wound at both time points using ImageJ [58].

Assessment of cell apoptosis
Cell apoptosis was measured using the Annexin V-FITC kit, following the manufacturer's instructions. Briefly, cells treated with the indicated chemical(s) for 48 h and then harvested by trypsinization. Cells were washed twice by centrifugation and re-suspended in PBS. Cells were then collected and re-suspended in 500 μL of the binding buffer. These cells were then stained with 5 μL of Annexin V-FITC and 5 μL of the propidium iodide staining solution for 15 min at room temperature in the dark. The percentage of Annexin V-FITC-and propidium iodide-positive cells was measured by flow cytometry (FACSAria, BD Biosciences, San Jose, USA).

Western blot analysis
All cells were collected and lysed in 200 μL radioimmunoprecipitation assay buffer with the protease inhibitor phenylmethylsulfonyl fluoride (100 mM) for 1 h on ice. Subsequently, the lysate was centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was collected and the total protein concentration was determined using a bicinchoninic acid kit (Thermo Fisher Scientific). Ten μg of total protein from each sample was separated by electrophoresis using 12% SDS-PAGE gels and transferred onto nitrocellulose membranes. The target proteins were measured using the primary antibodies (1:10,000) and their corresponding secondary antibodies (1:50,000), followed by development with an ECL Advance Western blotting detection kit. β-actin was detected as a loading control. Signals were developed on X-ray films following exposure to ECL advance luminescence. The intensity of each band was quantified using Quantity One 4.62 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Levels of phosphorylated proteins were determined as a ratio of total protein: p-Akt relative to Akt and p-ERK1/2 relative to ERK1/2.

Statistical analysis
All results were from three independent experiments and are expressed as mean ± standard deviation. Data were analyzed by one-way analysis of variance using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). A p < 0.05 was regarded as statistically significant.

Characterization of VSMCs
The isolated VSMCs were identified by α-SMA staining, a VSMC-specific marker. All cultures had comparable numbers of cells and more than 98% of the cells were α-SMA-positive ( Figure 1).

LIRA inhibited HG-induced proliferation of VSMCs
VSMCs treated for 48 h with HG showed a significant increase in cellular proliferation compared to controls (1.99 ± 0.13 vs. 1.25 ± 0.15; p < 0.01), and pretreatment with LIRA significantly attenuated this HG-induced VSMC proliferation (1.62 ± 0.15; p < 0.01 vs. HG) ( Figure 2). Treatment of VSMCs with LIRA alone had no effect on cell proliferation, as compared to the control cells. To exclude the possible influence of osmatic change on VSMCs during the HG treatment, mannitol was used in place of glucose, which had no effect on the proliferation of VSMCs.

LIRA inhibited HG-induced migration of VSMCs
The migration capacity of VSMCs was measured via Transwell migration and in vitro scratch assays.
A significantly greater number of cells migrated through the Transwell membrane in the HG group than in the control group (86.40 ± 4.22 vs. 51.40 ± 2.30 cells; p < 0.01). Treatment with LIRA or mannitol alone resulted in no significant increase in migration, as compared to the control group. LIRA pretreatment significantly reduced the number of VSMCs that migrated following HG treatment (72.40 ± 2.07 cells; p < 0.01 vs. HG) ( Figure 3A,B).
The scratch wound assay showed that treatment with HG resulted in significantly more wound healing than the control group (85.61 ± 6.36 vs. 37.25 ± 4.78%; p < 0.01). LIRA pretreatment was able to attenuate the effects of HG on VSMC migration (67.03 ± 5.61%; p < 0.05 vs. HG) ( Figure 3C or mannitol alone had no effect on scratch width, as compared to the control group.

LIRA attenuated the inhibitory effect of HG on VSMC apoptosis
Treatment with HG for 48 h significantly reduced the number of apoptotic VSMCs compared to controls (1.34 ± 0.12 vs. 8.67 ± 0.87%; p < 0.01). Pretreatment of VSMCs with LIRA in HG conditions resulted in a higher rate of apoptosis (3.44 ± 0.89%; p < 0.05 vs. HG) ( Figure 4A,B). Treatment with LIRA or mannitol alone resulted in similar numbers of apoptotic cells to the control sample.
To further investigate the mechanism of the HG-induced reduction in apoptosis, the expression of relevant apoptotic proteins was investigated by Western blotting (Figure 4C,D). The anti-apoptotic protein Bcl-2 was significantly upregulated (p < 0.01 vs. control) and the pro-apoptotic proteins cleaved caspase-3 and BAX were significantly downregulated after HG treatment (p < 0.01 vs. control). LIRA pretreatment reduced those effects by HG treatment, resulting in increased cleaved caspase-3 and Bax levels, and decreased Bcl-2 levels (p < 0.01 vs. HG) ( Figure 4C,D). PD98059, LY294002 or Exe(9-39) alone had no effect on cleaved caspase-3 expression (all p > 0.05 vs. control).

Discussion
Atherosclerosis is the most common cardiovascular complication of diabetes. Vascular remodeling is an important component in the development of atherosclerosis, and both are closely associated with pathologic changes in VSMCs, including changes in proliferation, migration, and apoptosis [59][60][61]. Previous studies have suggested that HG concentrations promote these altered cellular behaviors in VSMCs [15,16,22,[62][63][64]. However, these data are inconclusive and a mechanistic understanding is still lacking.

The involvement of PI3K/Akt and ERK1/2 signaling pathways in the HG-induced alteration of VSMC migration, proliferation, and apoptosis
Previous studies have shown that the ERK1/2 signaling pathway plays an important role in VSMC proliferation [65], and insulin [66,67] or HG [13,14] could facilitate proliferation via activation of this pathway. HG can also induce dynamic changes in VSMCs through activation of the PI3K/Akt pathway [16,[20][21][22][23]. Therefore, these pathways likely play a crucial role in the tissue damage induced by hypertension and atherosclerosis.
To explore whether these signaling pathways are involved in the HG-induced altered physiology of VSMCs, we first investigated whether HG concentrations altered (See figure on previous page.) Figure 4 Liraglutide (LIRA) attenuated the inhibitory effects of high glucose (HG) on apoptosis of cultured vascular smooth muscle cells (VSMCs). (A) Apoptosis was determined by staining with Annexin V-FITC (x-axis) and propidium iodide (y-axis). For each dot plot, the upper and lower right quadrants represent early apoptotic and late apoptotic cells, respectively. (B) Quantification of the apoptosis experiments. Total apoptosis refers to the sum of early and late apoptosis values. Results are expressed as mean ± standard deviation from three independent experiments; * p < 0.01 vs. control; # p < 0.05 vs. HG. (C) Protein expression of cleaved caspase-3 in VSMCs; * p < 0.01 vs. control; # p < 0.01 vs. HG; † p < 0.05, ** p < 0.01 vs. HG + LIRA. (D) Protein expression of Bcl-2 and Bax in VSMCs; * p < 0.01, ** p < 0.01 vs. control; # p < 0.01, ## p < 0.01 vs. HG. Protein expression was normalized to β-actin. The results from three independent experiments are presented as mean ± standard deviation.
ERK1/2 and PI3K/Akt activation. We then used specific inhibitors to block these signaling pathways. We show, in agreement with previous studies [13,15,17,23], that HG treatment of VSMCs significantly increases the phosphorylation of ERK1/2 and Akt. Furthermore, inhibition of these enzymes reduces HG-induced cell proliferation and migration. Pretreatment with ERK1/2 or PI3K inhibitors also effectively prevents the ability of HG to reduce VSMC apoptosis.
Thus, our data demonstrate that HG treatment of VSMCs alters cell migration, proliferation, and apoptosis via ERK1/2 and PI3K/Akt signaling pathways. These findings open a new avenue to further explore the underlying mechanism by which HG regulates VSMC cell migration in diabetic patients. Furthermore, our study identifies new research areas that may prove critical in deciphering the pathogenesis of, and provides a novel means to prevent, atherosclerosis in DM.
LIRA attenuates HG-induced cellular dynamic changes of VSMCs by activating GLP-1R and inhibiting PI3K/Akt and ERK1/2 signaling pathways GLP-1 analogues and receptor agonists have been used in the treatment of DM, increasing pancreatic protein content and mass via boosting S6 kinase phosphorylation and acinar cell mass [68]. LIRA, a GLP-1 analogue, effectively lowers the body weight of diabetic patients, which was associated with enhancing plasma cardiac natriuretic peptides levels [69]. Long-acting GLP-1 mimetics, such as domain antibodies to serum albumin, can sustain the activation of GLP-1R and reduce myocardial infarct size and injury in acute coronary syndrome [70]. Another GLP-1 agonist, exenatide, increases hydrogen sulfide, carbon monoxide, and nitric oxide production, and then reduces central arterial pressure in an animal model [71]. In carotid endarterectomy of diabetic patients, preoperational use of GLP-1R agonists significantly increases the expression of sirtuin-6, a protein involved in the inflammatory pathway of diabetic atherosclerotic lesions and plaque stabilization [72]. A prospective pilot clinical trial has demonstrated that LIRA decreases carotid intima-media thickness in patients with type 2 diabetes [73]. (D) Apoptosis rates were determined by flow cytometry, with the lower right and upper right quadrants representing early apoptotic and late apoptotic cells, respectively. These values were quantified, and the total apoptotic rate is the sum of the early and late apoptotic rates; * p < 0.01 vs. control; # p < 0.05 vs. HG; † p < 0.01, ** p < 0.05 vs. HG + LIRA. Data from three independent experiments are expressed as mean ± standard deviation.
In this study, we show that HG-induced VSMC alterations are prevented by treatment with LIRA. It is possible that in DM patients, LIRA protects the vascular system against atherosclerotic changes by similar mechanisms. Our results also demonstrate that LIRA by itself does not induce measurable physiologic changes in VSMCs, but only blocks HG-induced effects.
Previous studies have shown that GLP-1 protects cardiomyocytes and endothelial cells against cell apoptosis by activating ERK1/2 and/or PI3K/Akt [31,44,74]. However, our data demonstrate that LIRA inhibits this activation, observed as reduced phosphorylation levels in VSMCs. Moreover, synergistic effects are observed when LIRA pretreatment is combined with ERK1/2 or PI3K inhibitors. Although GLP-1 shows protections on cardiomyocytes and endothelial cells by activating ERK1/2 and/or PI3K/Akt [31,44,74], our data suggest that LIRA exerts the beneficial effects on VSMCs in part via inhibiting ERK1/2 and PI3K/ Akt pathways. As VSMCs play a distinct role in the development of diabetic atherosclerosis, the involvement of these signaling pathways may be cell-type specific, which requires further investigation.
Cardiomyocytes, endothelial cells, macrophages, and VSMCs all express the GLP-1R, which mediates the anti-inflammatory and anti-proliferation effects of GLP-1 [31,40,48]. To confirm that the effects of LIRA in HG-treated cells occur via GLP-1R activation, VSMCs were pretreated with Exe(9-39). Indeed, Exe(9-39) treatment abolished the beneficial effects of LIRA treatment, and reversed the suppression of Akt and ERK1/2 activation.

Conclusions
Collectively, this study shows that HG treatment facilitates migration and proliferation of VSMCs, inhibits cell apoptosis, and increases the phosphorylation of ERK1/2 and Akt. These effects are attenuated by LIRA pretreatment; LIRA treatment reduced HG-induced phosphorylation of ERK1/2 and Akt, suppressed cell migration and proliferation, and increased cell apoptosis. Moreover, inhibitors of ERK1/2 and PI3K prevent the damaging effects of HG on cultured VSMCs, suggesting that HG exerts injurious effects via these signaling pathways. Importantly, we also demonstrate that a GLP-1R antagonist can block the beneficial effects of LIRA on VSMCs exposed to HG. These effects were further enhanced by the coadministration of inhibitors of ERK1/2 or PI3K, demonstrating for the first time that LIRA treatment acts through GLP-1R to regulate these signaling pathways. As LIRA is an effective and safe therapeutic candidate, it may become a promising treatment option in the prevention of diabetic atherosclerosis.